Papillomaviruses (PV) have been linked to widespread, serious human diseases, especially carcinomas of the genital and oral mucosa. Tens of millions of women suffer from human papilloma virus (HPV) infection of the genital tract. Significant number of these women eventually develop cancer of the cervix. It has been estimated that perhaps twenty percent (20%) of all cancer deaths in women worldwide are from cancers which are associated with HPV. As many as 90% of all cervical cancer maybe linked to HPV.
Papillomaviruses also induce benign, dysplastic and malignant hyperproliferations of skin and mucosal epithelium (see, for example, Mansur and Androphy, (1993) Biochim Biophys Acta 1155:323-345; Pfister (1984) Rev. Physiol. Biochem. Pharmacol. 99:111-181; and Broker et al. (1986) Cancer Cells 4:17-36, for reviews of the molecular, cellular, and clinical aspects of the papillomaviruses).
HPV's are a heterogeneous group of DNA tumor viruses associated with hyperplastic (warts, condylomata), pre-malignant and malignant lesions (carcinomas) of squamous epithelium. Almost 70 HPV types have been identified, and different papillomavirus types are known to cause distinct diseases, c.f., zur Hausen, (1991) Virology 184:9-13; Pfister, (1987) Adv. Cancer Res., 48:113-147; and Syrjanen, (1984) Obstet. Gynecol. Survey 39:252-265. HPVs can be further classified either high risk (such as HPV type 16 [HPV-16] and HPV-18) or low risk (e.g, HPV-6 and HPV-11) on the basis of the clinical lesions with which they are associated and the relative propensity for these lesions to progress to cancer. For example, HPV types 1 and 2 cause common warts, and types 6 and 11 cause warts of the external genitalia, anus and cervix. HPV's can be isolated from the majority of cervical cancers, e.g., approximately 85 to 90% of human cervical cancers harbor the DNA of a high-risk HPV. Types 16, 18, 31 and 33 are particularly common; HPV-16 is present in about 50 percent of all cervical cancers.
The biological life cycle of the papillomaviruses appears to differ from most other viral pathogens. These viruses are believed to infect the basal or germ cells of the epithelium. Rather than proceeding to a lytic infection in which viral replication kills the cell, viral DNA transcription and replication are maintained at very low levels until higher strata of the epithelium are achieved. There, presumably in response to differentiation-specific signals, viral transcription accelerates, DNA synthesis begins and virion assemble occurs.
In HPV-positive genital cancers, the viral genomes are transcriptionally active, and two viral genes, E6 and E7, are invariably expressed. The high-risk HPVs encode two oncoproteins, E6 and E7, whose expression can extend the life span of squamous epithelial cells, which are a normal host cell for the papillomavirus. E6 and E7 together can result in the efficient immortalization of primary human cells (Hawley-Nelson et al., (1989) EMBO J., 8:3905-3910; Münger et al., (1989) J. Virol., 63:4417-4421; Watanabe et al., (1989) J. Virol., 63:965-969). E6 and E7 are expressed in HPV-positive cervical cancer-derived cell lines (Schneider-Gädicke et al., (1986) EMBO J., 5:2285-2292; Schwarz et al., (1985) Nature, (London) 314:111-114; Smotkin et al., (1986) Proc. Natl. Acad. Sci. USA, 83:4680-4684). Furthermore, although many genetic changes have occurred in cervical carcinoma cells, the continued expression of the viral oncoproteins is necessary since expression of antisense E6/E7 RNA results in decreased cell growth (von Knebel-Doeberitz et al., (1988) Cancer Res., 48:3780-3785). Similar to the transforming proteins of the other small DNA tumor viruses, simian virus (SV40) and adenovirus, the transforming properties of the E6 and E7 oncoproteins appear to be due at least in part to their capacity to functionally inactivate the p53 and the retinoblastoma (pRB) tumor suppressor proteins. The E6 proteins of HPV-16 and HPV-18 can complex and cause ubiquination-dependent degradation of p53 (Wemess et al., (1990) Science, 248:76-79; Schiffaer et al., Cell 75:495-505 (1993)). The high-risk HPV E7 proteins bind pRB more efficiently than the E7 proteins of low-risk HPVs (Barbosa et al., (1990) EMBO J., 9:153-160; Dyson et al., (1989) Science, 243:934-937; Münger et al., (1989) EMBO J., 8:4099-4015). It is believed that the functional inactivation of both p53 and pRB, and related regulatory pathways, by E6 and E7 are important steps in cervical carcinogenesis.
One characteristic of HPV-related carcinogenic progression is the frequent integration of the viral genome into the human chromosome in the cancer cells in a manner that results in the loss of expression of the viral E2 gene but maintains high levels of E6/E7 expression (Dürst et al., (1985) J. Gen. Virol., 66:1515-1522; Jeon et al., (1995) Proc. Natl. Acad. Sci. USA, 92:1654-1658). The product of the E2 open reading frame plays an important role in the complex transcriptional pattern of the HPV's. The E2 transcriptional activation protein (“the E2 protein”) is a trans-acting factor that activates transcription through specific binding to cis-acting E2 enhancer sequences in viral DNA (Androphy et al., (1987) Nature, 324:70-73), and has been shown to induce promoter expression in a classical enhancer mechanism (Spalholz et al., (1985) Cell 42:183-91). The E2 gene product exerts trans-regulatory effects in the upstream regulatory region (“LCR”) of the viral genome, disruption of E2 is thought to alter regulation of expression of E6 and E7 genes.
As with other transcription factors, the functions of the E2 proteins appear to be localized in discrete domains (Giri et al., (1988) EMBO J., 7:2923-29). The E2 amino terminus encompasses the transcriptional activation domain and binding site for the papillomavirus E1 replication protein. The E2 C-terminal domain is well conserved among the papillomaviruses, and contains the dimerization and DNA binding activities of E2. This domain sponsors sequence-specific interaction with DNA containing the sequence ACC(G)NNNN((C)GGT and represses the papillomavirus early promoter that drives expression of E6 and E7 (e.g., the P97 promoter of HPV16 and the P105 promoter of HPV 18). This is due to the position of E2 binding sites within the promoter: two of the four E2 binding sites within the P97 and P105 promoters immediately flank the TATA box and promoter proximal SP1 sites of these promoters, rendering them inaccessible to needed transcription factors.
The upstream regulatory region (or long control region (LCR)) is found immediately 5′ to the early genes of bovine papilloma viruses (BPV's) and other papillomaviruses. The LCR contains cis-acting regulatory signals, including an origin of DNA replication and several promoters that function in early transcription. The LCR also contains enhancer elements that activate transcription from the URR promoters and heterologous promoters (Sousa et al., (1990) Biochemica et Biophysica Acta 1032: 19-37).
The E2 enhancer elements are conditional, in that they stimulate transcription only when activated by a protein encoded by the E2 open reading frame (Romanczuk et al., (1990) J. of Virol. 64:2849-2859). Gene products from the E2 gene include the full-length transcriptional activator E2 protein and at least two truncated versions of the E2 protein BPV1 that function as transcriptional repressors. Transcriptional activation and repression of viral genes by E2 gene products constitute critical regulatory circuits in papillomavirus gene expression and DNA replication (reviewed in McBride et al., (1991) J. Biol. Chem. 266:18411-18414). Within the LCR, transcriptional regulation by the E2 protein depends on its direct binding to the nucleotide sequence 5′ACC(G)NNNN(C)GGT3′ (SEQ ID NO:9) (Androphy et al., supra; Dartmann et al., (1986) Virology, 151:124-30; Hirochika et al., (1987) J. Virol, 61:2599-606; P. Hawley-Nelson et al., (1988) EMBO J., 7:525-31; McBride et al., (1988) EMBO J., 7:533-39; McBride et al., J. of Biol. Chemistry 266:18411-1844 (1991); Demeret et al. J. Virol. 71:9343-9349 (1997); Desaintes et al. EMBO 16:504-514 (1997); Thierry et al. New Biol. 10:4431-4437 (1990); and Bernard et al. J. Virol. 63:4317-4324 (1989)).
European patent application 302,758 refers to the use of modified forms of E2 protein that bind to, and block, E2 binding sites on papillomavirus DNA without resulting in trans-activation. That application also refers to repression of E2 transcriptional activation through the use of DNA fragments that mimic E2 binding sites, and thus bind with E2 trans-activators, making them unavailable for binding to E2 sites on the viral DNA.
U.S. Pat. No. 5,219,990 describes the use of E2 trans-activation repressors which interfere with normal functioning of the native full-length E2 transcriptional activation protein of the papillomavirus. However, the E2 trans-activation repressors of the '990 patent are proteins that dimerize with the full-length native E2 protein to form inactive heterodimers, thus interfering with the formation of active homodimers comprising full-length native E2 polypeptides and thereby repressing papillomavirus transcription and replication. The E2 trans-activation repressors are described as fragments of the E2 polypeptide in which the dimerization function has been separated from its DNA binding function, e.g., the E2 trans-activation repressors includes at least the dimerization region, but less than the DNA binding domain, of the E2 polypeptide.